Using a molecular genetic approach, this project will focus mechanistically on alterations in developmental events that result in congenital malformations of the inner ear following prenatal alcohol exposure (Fetal Alcohol Syndrome). The ovedying hypothesis is that malformations of the inner ear subsequent to prenatal alcohol exposure are a result of decreased endogenous retinoic acid synthesis due to competition between alcohol and retinol for alcohol dehydrogenase enzyme pathways. Specific Aim 1 will examine the severity and range of malformations in the inner ear subsequent to prenatal ethanol exposure, or as a result of maternal vitamin A deficiency or excess. Additionally, alterations in gene pathways likely to result in dysmorphogenesis and/or cellular dysfunction will be analyzed using cDNA microarrays. The analysis will focus primarily on those genes known to be relevant for inner ear morphogenesis (e.g. FGF, EYA, GATA, BMP) and cell fate determination (e.g. Ngn1, Math1, Hes1, Hes5). Alternations in the spatio-temporal pattern of these genes will be analyzed using such methods as hierarchical and k-means clustering, self-organizing maps, and principal components analysis. Results will be verified using quantitative PCR (Q-PCR). Specific Aim 2 will confirm and extend the data from Specific Aim 1 by localizing expression of "critical genes" during inner ear development through the use of in situ hybridization and immunohistochemical techniques. Specific Aim 3 will directly test the suggested molecular interaction of ethanol and retinoic acid competition for common enzymatic pathways by using two different techniques (HPLC and a bioassay using retinoic acid responsive reporter cell lines). In a future RO1 submission, the identified candidate genes will be further analyzed in the teratogenic context using existing mutant and transgenic mouse strains (e.g. FGF10 null, ngn1 BAC) to probe for enhanced susceptibility.